1. Field of the Invention
This invention relates to the incorporation of nucleic acid into cellular systems, to vectors effecting such incorporation, and to microorganisms comprising such vectors.
More specifically, this invention relates to the novel plasmid pVE1, per se; its genome, or genetic component (DNA), entire and as fragments; and its derivatives (deletion and hybrid variants thereof) which are useful as cloning vectors into organisms, such as bacteria, including in particular plasmid pVE1 host strains, for example, Streptomyces avermitilis MA4990, S. lividans, or the like. The resultant modified cells are novel and have utility either as a means of producing the foreign nucleic acid and/or its products through replication of the cells, or through the imparting of valuable properties to the cells by virtue of the presence of the foreign nucleic acid therein.
2. Brief Description of the Prior Art
The use of plasmids as cloning vectors is a well established procedure. However, the application of these techniques to Streptomyces is relatively new.
DNA cloning with plasmid vectors and Streptomyces has been done primarily in S. lividans and S. coelicolor A3(2). SCP2, a low copy number plasmid of 31 kb, was used to clone the gene for methylenomycin resistance [(1) Bibb, M. J., Schottel, J. L., Cohen, S. N., 1980. "A DNA cloning system for interspecies gene transfer in antibiotic producing Streptomyces," Nature 284: 526-531]; and to clone S. coelicolor genes which complemented auxotrophic mutations [(2) Thompson, C. J., Ward, J. and Hopwood, D., 1982, "Cloning of antibiotic resistance and nutritional genes in Streptomyces," J. Bacteriol, 151: 668-677]. Genes encoding resistance to neomycin, thiostrepton, and viomycin have been cloned into the SLP1 plasmid family, a group of low copy number plasmids derived from the S. coelicolor A3(2) chromosome [(3) Bibb, M. J., Ward, J. M., Kieser, T., Cohen, S. N. and Hopwood, D., 1981, "Excision of chromosomal DNA sequences for Streptomyces coelicolor forms a novel family of plasmids detectable in Streptromyces lividans, " Mol. Gen. Genet., 184: 230-240; (4) Thompson, C. J, Ward, J. M. and Hopwood, D. A., 1980, "DNA cloning in Streptomyces: Resistance genes from antibiotic producing species," Nature 286: 525-527; Thompson, C. J., Ward, J. M. and Hopwood, D. A., 1982, supra]. The genes for neomycin, thiostrepton and viomycin resistance (aph, tsr and vph, respectively) were also cloned into a high copy number, broad host range plasmid, pIJ101, obtained from S. lividans ISP 5434 [(5) Kieser, T., Hopwood, D. A., Wright, H. M. and Thompson, C. J., 1982, pIJ101, "A multi-copy broad host range Streptomyces plasmid: Functional analysis and development of DNA cloning vectors," Molec. Gen. Genet. 185: 223-238]. This plasmid has also been isolated from S. coelicolor ATCC 10147 [(6) Pernodet, J. and Guerineau, M., 1981, "Isolation and physical characterization of Streptomyces plasmids," Mol. Gen. Genet. 182: 53-59]. SCP2, SLP1 and pIJ101 are all self-transmissible plasmids. When a strain containing one of these plasmids is plated on a plasmid-free strain, a small zone of growth inhibition, known as a pock, is formed in the lawn around the plasmid-containing strain. Plasmid DNA is introduced into Streptomyces by polyethylene glycol (PEG) mediated transformation of protoplasts [(7) Okanishi, M., Suzuki, K. and Umezawa, H., 1974, "Formation and reversion of Streptomyces protoplasts: Cultural conditions and morphological study," J. Gen. Microbiol. 80: 389-400; (8) Bibb, M. J., Ward, J. M. and Hopwood, D. A., 1978, "Transformation of plasmid DNA into Streptomyces at high frequency," Nature, 274: 398-400].
Bacteriophage have also been used as cloning vectors in Streptomyces [(9) Isogai, T., Takahashi, H. and Saito, H., 1981, "Actinophage R4 as a DNA cloning vector in Streptomyces," J. Gen. Appl. Microbiol. 27: 373-379; (10) Suarez, J. E. and Chater, K. F., 1980, "DNA cloning in Streptomyces: A bifunctional replicon comprising pBR322 inserted into a Streptomyces phage," Nature, 286: 527-529]. A review of methods and currrent progress in Streptomyces cloning can be found in (11) Chater, K. F., Hopwood, D. A., Kieser, T. and Thompson, C. J., 1982, "Gene cloning in Streptomyces," Curr. Top. Microbiol. Immunol. 96: 69-95.
Hybrid replicons which function in E. coli and Streptomyces have been constructed by linking the E coli plasmid pBR322 to Streptomyces phage .phi.C31 (Suarez, J. E. and Chater, K. F., 1980, supra), to a derivative of SLP1 (Chater, K. F., Hopwood, D. A., Kieser, T. and Thompson, C. J., 1982, supra), and to a Streptomyces espinosus plasmid pUC6 [(12) European Patent Application 0035914A2; No. 81301009.7; published Sept. 16. 1981] and also by joining pACYC177 or pACYC184 to a derivative of SLP1 [(13) Schottel, J. L., Bibb, M. J. and Cohen, S. N., 1981," Cloning and expression in Streptomyces lividans of antibiotic resistance genes derived from Escherichia coli," J. Bacteriol. 146: 360-368].
A great variety of plasmids have been observed in many different Streptomyces and may have the potential to be developed into cloning vectors. For example, see the following:
(14) Chung, S. T., 1982, "Isolation and characterization of Streptomyces fradiae plasmids which are prophage of the actinophage .phi.SF1," Gene 17: 239-246.
(15) Manis, J. J. and Highlander, S. K., 1982, "Partial characterization of a small, multi-copy plasmid from Streptomyces espinosus and the derivation of a high copy-number deletion mutant," Gene 18: 13-20.
(16) Kirby, R., Lewis, E. and Botha, C., 1982," A survey of Streptomyces species for covalently closed circular (ccc) DNA using a variety of methods, "FEMS Microbiol. Let. 13: 79-82.
(17) Omura, S., Ikeda, H. and Tanaka, H., 1981," Extraction and characterization of plasmids from macrolide antibiotic-producing Streptomycetes," J. Antibiotics 34: 478-481.
(18) Yi-Guang, W., Davies, J. and Hutchinson, C. R., 1982, "Plasmid DNA in the erythromycin producing microorganism Streptomyces erythreus NRRL 2338," J. Antibiotics 35: 335-342.
(19) Toyama, H., Hayashi, E., Nojiri, C., Katsumata, K., Miyata, A. and Yamada, Y., 1982, "Isolation and characterization of small plasmids from Streptomyces," J. Antibiotics. 35 369-373.
(20) Okanishi, M., Manome, T. and Umezawa, H., 1980, "Isolation and charaterization of plasmid DNAs in actinomycetes," J. Antibiotics 33: 88-91.
(21) Hayakawa, T., Otake, N., Yonehara, H., Tanaka, T. and Sakaguchi, K., 1979, "Isolation and characterization of plasmids from Streptomyces," J. Antibiotics 32: 1348-1350.
(22) UK Patent Application G.B. 2045253A; No. 8007080; published Oct. 29, 1980 (pUC1).
(23) UK Patent Application G.B. 2045252A; No. 8007079; published Oct. 29, 1980 (pUC2).
(24) UK Patent Application G.B. 2044773A; No. 8007077; published Oct. 22, 1980 (pUC3).
(25) UK Patent application G.B. 2043652A; No. 8007158; published Oct. 8, 1980 (pUC6).
(26) UK Patent Application G.B. 2046272A; No. 8011000; published Nov. 12, 1980 (pUC7).
(27) UK Patent Application G.B. 2045251A; No. 8007078; published Oct. 22, 1980 (pUC8).
(28) UK Patent Application G.B. 2045254A; No. 8007081; published Oct. 29, 1980 (pUC9).
(29) European Patent Application 0038156A2; No. 81301482.6; published Oct. 21, 1981 (pUC10).
(30) European Patent Application 0035914A2; No. 81301009.7; published Sept. 16, 1981 (pUC1012 and pUC1013).
(31) European Patent Application 0020251A2; No. 80400722.7; published Dec. 10, 1980 (hepatitis B plasmid vector).
(32) UK Patent Application G.B. 2031434A; No. 7928156; published Apr. 23, 1980 (Chimeric proteins to make hepatitis B antigen).